Mechanisms of Efficient Boundary-Pairing: Investigating the Context-Dependent Significance of motif8 in Long-Distance even-skipped Boundary Interactions

Abstract

Boundaries, cis-regulatory elements that demarcate topologically associating domains, exhibit insulator function and dictate specific enhancer-promoter interactions by directing physical looping. Under the boundary-pairing model, loop topologies are determined by specific, orientation-dependent boundary pairing which can occur across long distances. The specific mechanisms facilitating boundary pairing are not yet elucidated. A computational model has predicted the ten base pair sequence, motif8, to be significant for efficient pairing by one Drosophila melanogaster even-skipped boundary (neighbor of homie, nhomie), but not the other (homie). Adapting a dual-reporter transgene system by Fujioka et al. (2016) and Bing et al. (2024), we assessed the significance of motif8 to efficient, long-distance pairing by transgenic boundary constructs with the endogenous eve locus in vivo. To investigate differences across boundary compositions, we employed variations of transgenes containing copies of endogenous nhomie, homie, or a shortened nhomie. We observed decreased reporter expression with a motif8 mutation in transgenic nhomie indicative of reduced efficient, long-distance pairing. Contrastingly, a lack of significant decreases and low baseline reporter expression was observed with homie or shortened nhomie variations, suggesting a context-dependent significance for motif8. Our preliminary investigation of CG4854, an uncharacterized protein predicted to associate with motif8, indicated ubiquitous expression in early-stage embryos. A GFP:CG4854 proxy for endogenous expression also suggested low levels of DNA-association. This assessment of motif8 furthers the boundary-pairing model by highlighting the significance of intra-boundary compositions to inter-boundary interactions, adding to our understanding of the mechanisms dictating chromatin architecture.